This invention relates to a composition comprising a Bowman-Birk Inhibitor Concentrate (BBIC) product prepared from the soybean and uses thereof. This BBIC product has been shown to exhibit surprisingly high inhibitory activity against the malignant transformation of cells under certain conditions and its administration to affect various forms of cancer development. It is now believed that BBIC inhibits the malignant transformation of cells by destroying pre-malignant tissue by the induction of high levels of proteolytic activity.
It has been shown that the enzyme-inhibitor described by Bowman, Proc. Soc. Exptl. Med. 1946, 63, 547 and Birk et al., Bull. Res. Council Israel 1962, Sec. 1, 11, 48 and Biochim. Biophys. Acta 1963, 67, 326, and subsequently referred to as the Bowman-Birk Inhibitor (BBI), possesses certain physiological activity that prevents, or at least greatly reduces, radiologically or chemically induced malignant transformation of cells in culture and in experimental animals.
Yavelow et al., Proc. Natl. Acad. Sci. USA 1985, 82, 5395-5399, reported that a crude soybean extract, if defatted with acetone, effectively blocked cell transformation in vitro. These observations, with epidemiological data, suggested BBI as a putative dietary anticarcinogen, particularly with respect to colon cancer.
Weed et al., Carcinogenesis 1985, 6, 1239-1241, disclose that an extract of soybeans containing the Bowman-Birk protease inhibitor added to the diet of dimethylhydrazine (DMH)-treated mice resulted in a significant suppression of adenomatous tumors of the colonic mucosa. DMH-induced colon cancer in mice is generally regarded as an excellent animal model for the human disease, with carcinogen treatment inducing adenocarcinomas of the colon and rectum which are similar to the tumors arising in the human colon suggesting the possibility that a dietary additive of the sort studied might confer some protection against the development of human colon cancer without undesirable side effects. The BBI extract and methods for its preparation were as described by Yavelow et al., Cancer Res. 1983, 43, 2454-2459; Proc. Natl. Acad. Sci. USA 1985, 82, 5395-5399 and Hwang et al., Biochim. Biophys. Acta 1977, 495, 369-382.
Messadi et al., JNCI 1986, 76,447-452 demonstrated that a soybean extract containing the protease inhibitor BBI suppresses 7, 12-dimethyl-benz[a]anthracene (DMBA)-induced carcinogenesis in the hamster cheek pouch. This oral cancer model has the same histopathology, growth pattern and precancerous lesions as the most common form of human oral cancer, squamous cell carcinoma. It was shown in this study that hamster cheek pouch carcinogenesis can be inhibited by BBI and suggested that human oral carcinogenesis might respond to BBI in a comparable manner. The BBI preparation used in this study was a crude extract of the inhibitor prepared as described by Yavelow et al., Proc. Natl. Acad. Sci. USA 1985, 82, 5395-5399.
Baturay et al., Cell Biology and Toxicology 1986, 2, 21-32 disclose that a BBI preparation, wherein a crude soybean extract is defatted with acetone, suppresses radiation and chemically induced transformation in vitro, with or without enhancement by the co-carcinogen, pyrene. Yavelow et al., 1985, show that either pure BBI or the BBI extract prepared in accordance with their methods suppresses radiation induced transformation in C3H10T1/2 cells. Kennedy et al., 1984, report that either pure BBI or the BBI extract prepared in accordance with their method reduce the levels of chromosome abnormalities in cells of patients with Bloom's syndrome (a genetic disease in which the high levels of chromosome abnormalities are thought to predispose the patients to a higher than normal cancer incidence). Still, other studies suggest that soybean-derived protease inhibitors can have suppressive effects on skin, breast and liver carcinogenesis in vivo.
Kennedy et al. in Anticarcinogenesis and Radiation Protection, edited by Cerutti et al., Plenum Pub. Co., pp. 285-295 (1987), disclose that BBI suppresses carcinogenesis in various systems using a crude BBI extract prepared by defatting soybeans with acetone. Their results suggested that very low concentrations of BBI-type protease inhibitor preparations would be effective as chemopreventive agents for colon cancer. There was no evidence to suggest that the use of protease inhibitors as chemopreventive agents would be complicated by possible toxicity problems.
St. Clair et al., Cancer Res. 1990, 50, 580-586, report that the addition of 0.5% or 0.1% semi-purified BBI to the diet of DMH-treated mice resulted in a statistically significant suppression of angiosarcomas and nodular hyperplasia of the liver and colon carcinogenesis. The results of this study also indicate that BBI, included as 0.5% of the diet or less had no adverse effect upon the health of the mice but had the capacity to suppress liver and colon carcinogenesis.
Perlmann et al., Methods in Enzymology 1970, 19, 860-861, have described an elaborate method for obtaining BBI from a defatted soybean extract.
U.S. Pat. No. 4,793,996 (Kennedy et al.) discloses a process comprising treating soybeans with acetone, followed by ethanol extraction and acetone precipitation for obtaining BBI. The soybeans may be defatted prior to acetone treatment. In addition, BBI may be further purified by conventional techniques. Kennedy et al. discovered that in the conventional process for preparing BBI from soybeans, a factor remained which adversely affected the ability of BBI to inhibit the malignant transformation of cells. If the factor was removed, the resulting BBI product was capable of inhibiting the malignant transformation of cells. It was found to be possible to remove this factor by treating the soybeans with acetone prior to the ethanol extraction step taught by Perlmann et al.
Kennedy et al. teach that it is unnecessary to carry out a procedure requiring complete purification of the extract to the point where the product contains only a single protein. Instead, they found it effective to stop the purification procedure at a point where a crude inhibitor extract is obtained. This crude extract is itself edible and can be used as an inhibitor of malignant transformation of cells, for example, by oral ingestion. Kennedy et al. disclose a process for preparing a crude soybean extract containing an inhibitor of malignant cell transformation which comprises defatting soybeans and extracting said inhibitor from said defatted soybeans. The improvement comprises defatting said soybeans by bringing them into contact with at least an equal weight of acetone and, thus, producing a crude inhibitor extract having greatly increased effectiveness.
The prior art has also described concentration of BBI from soybean solubles by centrifugation and ultrafiltration, and further purification by acetone precipitation. The separation of soybean solids from hexane-extracted soy flour/flakes in a commercial soy protein concentrate process is well known. However, the BBIC product of this invention produced by these steps alone, i.e., without the use of an aqueous alcohol extraction, is both novel and unexpected as this BBIC product is unexpectedly more effective in suppressing cancer development.
Furthermore, production of this BBIC product uses less solvent and thus is a more economical and safer process. Producing waste-solvent streams containing a mixture of alcohol-water-acetone requires very complex and expensive distillation equipment, which is eliminated in the present invention. It has also been discovered that ultrafiltration is much more efficient than dialysis; one single step of ultrafiltration can remove more solids than three days of dialysis. After purification, most of the examples of the present invention employ spray-drying, which is much faster and hence, more economical than the lyophilization described in the prior art. Unexpectedly, spray-drying has no effect on BBI recovery, as measured by Chymotrypsin Inhibitor (CI) content, used as an indicator for the presence of BBI.